EM visualization of Pol II genes in Drosophila: most genes terminate without prior 3' end cleavage of nascent transcripts.

Abstract

Transcription termination by RNA polymerase II (Pol II) on most mRNA-encoding genes is dependent on transcription through a functional poly(A) signal. One model to explain this dependence predicts co-trancriptional cleavage of RNA at the poly(A) site. Electron microscopic (EM) visualization was used to investigate the in vivo frequency of transcript cleavage prior to termination. Over 100 unidentified Drosophila Pol II-transcribed genes were analyzed. Although some genes exhibited cleaved transcripts near their 3' ends, and some had a lower polymerase density at their 3' end relative to the rest of the gene, the majority of genes (64%) had uncleaved transcripts and no change in polymerase density at the 3' end, consistent with release of full-length transcripts at a discrete site. Thus, in Drosophila, cleavage at the poly(A) site sometimes occurs co-transcriptionally, but does not appear to be a prerequisite to termination. Next, two components of the polyadenylation complex were immunolocalized on polytene chromosomes and were found to differ in distribution both qualitatively and quantitatively. The EM results indicate that co-transcriptional recognition of the poly(A) signal, which is required for termination, does not equate with co-transcriptional cleavage, and the immunofluorescence results suggest that this may be due to incomplete or nonstoichiometric assembly of the polyadenylation machinery on nascent transcripts.